Paper Thesis

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Acute Toxicity of Percutaneously Absorbed Malathion, anOrganophosphate, on Bufo sp. Larvae

Mej Amm M. BatoonNatural Sciences and Mathematics Division

UP in the Visayas, Gorordo Ave., Lahug Cebu City 6000

Abstract

Anurans are important bioindicators for environmental toxinsdue to their biphasic lifestyle, permeable skin and sensitivity tochemical toxins, such as malathion. This experiment studied theacute toxicity of 5 concentrations (1 ppm, 5 ppm, 10 ppm, 15 ppm and20 ppm) of technical grade malathion through percutaneousabsorption in Bufo sp. field stage IV larvae. The calculated values ofLC50 from 9th-12th day ranged from 6 to 13 ppm in a decreasing

pattern, showing that levels of toxicity ofBufo sp. field stage IV larvawith malathion increase with constant exposure. Mortality was foundto be dosage dependent (R2 = 0.9194). Exposure also producedabnormalities in morphology including: axis deformities in the headand tail and the presence of a bulge on the lower right abdominalregion. Abnormalities such as tail curvature and head bending weredosage dependent (R2 = 0.9431 and 0.7876, respectively) signifyingpositive relationships with increase malathion concentration. Tailcurvature was significantly greatest in the highest concentration(P


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Important toxins introduced to the environment are pesticides, which

are chemicals used in agriculture and households to remove pests from crops

and many insects. Organophosphate pesticides are the most widely used

class of insecticides in the world. These pesticides have numerous

agricultural, horticultural, industrial, and medical applications spanning every

conceivable insecticide, acaricide, and nematocide in the market (Racke,

1992; Diana et al., 2001).

Malathion is one of the earliest organophosphate developed and

introduced in 1950 and has been used to kill insects on many types of crops

since this time (Hunter and Barker, 2003). In the Philippines, malathion is the

second most common pesticides used for crops (Dioquino, 2002). Malathion

is also used to control mosquitoes, flies, household insect, animal parasites

(ectoparasites) and head and body lice. However, studies have shown that

malathion in certain concentrations can cause adverse effects to nontarget

species, such as frogs and toads, found in areas where pesticides spraying

usually occur (Fordham et al. 2001; Gilbertson et al. 2003; Taylor et al. 1999;

Giles and Roberts, 1970).

In aquatic habitats, malathion has been detected at concentrations up

to 0.6 mg/L. Although, malathion does not persist in the environment with its

half-life of only 6 days up to several weeks. Degradation depends on

environmental conditions such as pH, moisture, presence of bacteria and

light. Despite malathions rapid degradation, even brief exposure can alter the

development of non-target animals, particularly aquatic vertebrate embryos

(Cook et al., 2005). Concentrations as small as 1 ppm can already cause

adverse effects to frog larvae after 4 days of exposure (Bulletin of

Environmental Contamination Toxicology, 31, 170-176, 1983). In addition,

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malathion degradation also results to compounds more toxic than malathion

(Effect of Impurities on the Mammalian Toxicity of Technical Malathion and

Acephate. Journal of Agricultural Food Chemistry, 25 (4): 946-953, 1977).

Most studies on organophosphates are focused on chronic toxicities on

the development of anuran larvae to adult. The results of which are

deformities such as extra or missing limbs or digits. This study, however,

focuses on the acute toxicity of absorbed malathion on Bufo sp. larvae, which

is the most common anuran species found in the Philippines. Acute toxicity

studies on malathion were usually done on mammals, such as rats and

rodents. Amphibian toxicity studies were more on Gosner stage 25, where

mouthparts are prominent and the spiracle is visible. This study followed the

simplified 8-stage of tadpole development where each stage or field stage

includes several Gosner stages with similar developmental occurrence, e.g

limb bud formation under field stage 2 or Gosner stages 26-30. This study

involved field stages 2 and 3, which includes Gosner stages 26-30 and 31-35,

respectively, or when the limb bud and toe development occurs.

Review of Literature

What is Malathion?

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Malathion is a manufactured product (molecular weight: 330.3503) that

belongs to a class of insecticides known as organophosphate (OPs). Its

structure containing a P=S bond and another S attached with an alkyl group

places it in the subclass of OPs known as phosphorothionothiolates (Figure 1)

(Masicotte, 2001).

Malathion pesticides usually come in two forms: a purified form (which

is approximately 99% malathion) of colorless liquid and a technical-grade

solution (which contains approximately 96.5% malathion) with a brownish-

yellow liquid and garlic-smell). It is available under different product names

including Celthion, Cythion, Dielathion, El 4049, Emmaton, Exathios, Fyfanon

and Hilthion, Karbofos and Maltox. It is usually available in emulsifiable

concentrate, wettable powder, dustable powder and ultra low volume liquid

formulations. Most common solvent used for technical grade malathion

include xylene. Application is usually done by spraying over target areas.

Application in pets usually includes dipping of the animal into a solution of

malathion.

Figure 1. Molecular structure of Malathion

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Malathion toxicity

EPA toxicity tests classified malathion under toxicity class III as a

slightly toxic compound. Malathion interferes with the nervous system function

by inhibiting acetylcholine esterase, an enzyme that degrades acetylcholine

signals so the next nerve impulse can be transmitted across the synaptic gap,

thereby paralyzing and killing insects. Studies have shown that malathion is

carcinogenic and has been linked with increased incidence of leukemia in

mammals. Chronic effects of malathion includes: delayed mutagen and

teratogen, delayed neurotoxin, allergic reactions, behavioral effects, ulcers,

eye damage, abnormal brainwaves and immunosupression (Effect of

Impurities on the Mammalian Toxicity of Technical Malathion and Acephate.

Journal of Agricultural Food Chemistry, 25 (4): 946-953, 1977).

In humans, exposure to high amounts of malathion in the air, water, or

food may cause difficulty in breathing, chest tightness, vomiting, cramps,

diarrhea, watery eyes, blurred vision, salivation, sweating, headaches,

dizziness, loss of consciousness, and death (DuBois, 1971).

Degradation of malathion

Malathion is easily degraded in the environment; a reason why it is one

of the most popularly used pesticide. Degradation can be through different

pathways, such as: volatilization, photolysis, hydrolysis, and microbial

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degradation (Racke, 1992; Massachusetts Department of Agricultural

Resources, 2005). Conditions in aquatic environments can also enhance or

decrease the rate of degradation. Hydrolysis (halflife=6 days) degradation of

malathion can be enhanced by high pH, high temperatures and ultraviolet

radiation (Chambers, 1992). Oxidation or desulfuration (oxidation of malathion

P=S to P=O oxon intermediate) can produce two metabolites, malaoxon and

0,S,S-trimethyl phosphorothioate, which is respectively 60 times and 500

times more toxic than malathion. Malaoxon, however, has lower lyphophilic

property, therefore percutaneous absorption is less likely to occur (Tsuda et

al., 1997).

Percutaneous absorption in anurans

Percutaneous absorption of xenobiotics, or chemicals not naturally

occurring within the body, such as malathion is an important route for

anthropogenic environmental exposure in amphibians considering the

potential for extended contact with this compounds in aquatic environments

where they are found (Wallace, 1992; Taylor 1999a,b; Johnson et al. 2000;

Fordham 2001; Relyea 2004). Paracellular, transcellular, and

transappendageal pathways are three routes whereby xenobiotics are

percutaneously absorbed (Riviere, 1999). Paracellular involves transport

through intercellular lipids. Amphibians also posses this kind of morphology.

Trancellular pathway involve molecules transfer through cells, as well as

intercellular lipid matrix. While transappendageal pathway aids in cutaneous

absorption through transport involving hair follicles and other adnexa. This

type of cutaneous absorption contributes to the high bioaccumulation of OP

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pesticides in amphibian skin (Hall and Kolbe, 1980; Ling, 1990). The

transappendageal pathway aids in cutaneous absorption through transport

involving hair follicles and other adnexa, which are appendages of an organ

e.g hair follicle of skin. However, amphibians do not possess hair follicles

contains a significant distribution of cutaneous serous and mucous glands as

sites for absorption (Goniakowska-Witalinska and Kubiczek, 1998; Green,

2001).

Anuran skin maintains a bifacial cell system with respect to solute

permeability which results to a unidirectional flow of solutes from exterior to

interior. This is due to the depolarization of the exterior cell surface of skin

epithelium and not in the basal surface, which therefore results to a higher

permeability to the contaminant in the former and lower permeability in the

latter (Ling, 1990).

Hypotheses

Based on the known impacts of malathion and readings from literature,

it was hypothesized that:

1) Malathion at varying concentrations will kill fifty percent (50%) of the

population of the test Bufo sp. larvae

2) Number of deaths will vary significantly across treatments and from

control group.

3) Mortality ofBufo sp. larvae will be dose dependent.

4) Malathion will cause a significantly higher degree of deformities (such

as, deformed body axis and increase liver size) in Bufo sp. larvae.

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5) The increase of degree of deformities (body axis and liver size) will

increase with concentration.

Objectives

This study was conducted to determine the acute toxicity of malathion

on a population of tadpoles collected from Family Park, Talamban, Cebu.

Specifically it aimed:

1) to identify the LC50 at different exposure time.

2) to determine if the number of deaths will vary significantly across

treatments and from the control group.

3) to determine if mortality ofBufo sp. larvae will be dose dependent.

4) to identify and determine if degree of deformities associated with

exposure to malathion is significantly different across treatments and

the control.

5) to determine if the degree of deformity (body axis and liver size) will

increase with concentration.

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MATERIALS AND METHODS

The acute toxicity test for malathion to tadpoles were performed within

the Biology laboratory of the University of the Philippines Cebu College Arts

and Sciences Building.

Sample Collection

Bufo sp. larvae were collected from a clean permanent pond located in

Family Park, Talamban, Cebu. Only a single collection of samples was

performed for all treatments. Tadpoles collected were of similar sizes and

similar developmental stage. Basing from previous sampling, it was observed

that new eggs were not laid before tadpoles fully developed to adult frogs.

This is due to the tendency of tadpoles to cannibalize on small and weaker

tadpoles. Therefore, samples collected were of similar age and come from the

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same egg clusters. Sample collected belonged to field stage 4 larval stage

[36th 40th Gosner (1960) stages]. Prior to experimentation, the samples were

subjected to 24-hour acclimatization.

Preparation of Treatment Set-up

Prior to the conduct of final experiment, several test runs were

performed to determine acceptable conditions, such as: the concentration

range of malathion, type of medium (natal water or distilled water), condition

of the larvae (starved or fed) and type of vessel (petri dish or 5-L jar) for the

acute toxicity test.

Acceptable concentrations included: 1, 5, 10, 15 and 20 ppm, which

were prepared by serial dilution of technical grade malathion (570 g/L

malathion, 80 g/L emulsifier and 350 g/L xylene). Distilled water was preferred

over natal water as medium since the latter could contain dissolved

substances that could potentially affect the results of the experiment. The

volume of the solution used was 300 ml, since previous test runs had shown

that higher volumes with the same concentration contain higher amounts of

dissolved malathion resulting to higher exposure, while much lesser volumes

limit available oxygen required for respiration. The vessel found appropriate

for the each set-up was a 5-L jar, which had greater volume-capacity and

bigger diameter at the bottom than Petri dish, in order to allow more space for

the larva to swim and avoid stress due to crowding.

Three set-ups were made per treatment with 10 tadpoles, which served

as individual cases rather than replicates, per set-up. Therefore, 30 individual

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cases were used per treatment including the control groups, which only

contained 300 ml distilled water per set up.

Throughout the experiment, external conditions were controlled: lights

were turned on and off with a 12:12 hour ratio on a daily basis for

photoperiodism. The larvae were starved throughout the experiment to limit

exposure through percutaneous absorption and to avoid exposure through the

gut, which was found to be more lethal and biological factors such as

degradation of food also result to mortality of the larvae in both treated and in

the control as observed in previous test runs.

The experiment lasted until mortality was observed in the control,

which was after 12 days.

Acute Toxicity Testing

The static toxicity test was patterned after Sayim et al. (2005).

Tadpoles were observed for occurrence of mortality and malformations at the

end of every 24 hour period throughout the course of the experiment. Dead

animals were removed during each observation.

Deformities including degree of tail curvature and increased liver size,

were measured using the profile projector. Behaviors, including swimming and

balance of larvae were also noted.

Data Analysis

LC50and 95% confidence interval

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Mortality data from the replicate samples from each malathion

concentration were pooled prior to calculating LC50 and 95% confidence

intervals. The 96-hour LC50 and 95% confidence interval were determined

using Probit analysis with SPSS version 10.0 for windows.

Tail curvature, head bending and liver size

Mean of tail curvature was compared per treatment and with the control

if they varied significantly using ANOVA SPSS version 10.0 for windows. The

change in degree of tail curvature in response to increasing concentration of

malathion were analyzed using linear regression.

Similar procedure was done in the analysis of other abnormalities, such

as degree of head bending and increase liver size.

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RESULTS

Mortality

One hundred percent (100%) mortality in the population of Bufo sp.

larvae was observed after 48 hours (2 days) of exposure to 20 ppm

concentration of malathion. Fifty percent (50%) mortality was attained after

144 hours (6 days) of exposure to 15 ppm malathion and after 264 days (11

days) of exposure to 10 ppm malathion. Death occurred simultaneously with

the lower concentrations but did not reach to 50% of the population at the end

of the observation period (Figure 2). Meanwhile, no mortality occurred in the

control group throughout the course of the experiment.

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0

20

40

60

80

100

120

0 1 5 10 15 20

Concentration (ppm)

Mortality

(%)

Figure 2. Percent mortality of Bufo sp. in different concentrations of

malathion.

Larvae mortality was observed to be dose-dependent (Figure 3). Using

Linear regression, the R2 value obtained was 0.9194, signifying that the

increase in mortality is dependent and related to the increase in the

concentration of malathion.

R2

= 0.9194

0

20

40

60

80

100

120

0 5 10 15 20 25

Concentration (ppm)

Mortality(%

Figure 3.Bufo sp. field stage 4 larvae mortality throughout 12-day exposure

period to malathion.

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The calculated LC50 values for malathion for 9-12 days of exposure is

displayed in Table 1. As seen in Table 1, the concentration to achieve LC50 in

the experiment is decreasing as the length of exposure was increased.

Table 1. Lethal concentrations (LC10, LC50, and LC90) in ppm for Buffo sp.larvae exposed to malathion

DayLC10

(95% CI)

LC50

(95% CI)

LC90

(95% CI)P value

95.96889

(-52.98967-10.72630)

13.47137

(8.67738-70.22988)

20.97385

(14.53161-185.54629)0.026

103.39752

(-15.83596-7.56189)

12.13965

(8.01651-27.44735)

20.88179

(14.57982-64.62197)0.086

112.04066

(-0.70704-3.83990)

9.58642

(8.13695-11.35248)

17.13217

(14.70630-21.13970)0.477

120.03329

(-8.43267-3.12100)

6.42104

(3.40900-10.03471)

12.80879

(9.41705-22.78203)0.141

Occurrence of Degree of Deformities

Observable deformities in the tadpole exposed to different

concentrations of malathion included curved tail in dead larvae, bent head

resembling the structure of a golf club and a bulge in the right abdominal

region, which became more prominent in higher concentrations (Figure 4).

Although most of these deformities were found in the tadpoles exposed to 15

ppm of malathion, only tail curvature was found in tadpoles that were exposed

to 20 ppm, which died after 48 hours of exposure.

Mean tail curvature ranged from 2 at 5 ppm to 12 at 20 ppm. These

values were found to differ significantly among treatment groups and from the

control with tail curvature at 20 ppm as significantly highest among the

treatment groups. However, the degree of tail curvature at 20 ppm did not

significantly differ with degree of tail curvature at 15 ppm, but was

significantly higher (p


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ppm, 5 ppm and 10 ppm) and the control. Degree of tail curvature in 15 ppm

differed significantly (p


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R2

= 0.9431

0

2

4

6

8

10

12

14

0 5 10 15 20 25

Concentration (ppm)

Angle(degrees)

Figure 5. Tail curvature of Bufo sp. field stage 4 larvae after malathionexposure.

R2

= 0.7876

0

1

2

3

4

5

6

7

8

9

0 5 10 15 20

Concentration (ppm)

Angle(degrees)

Figure 6. Angle of head bending of Bufo sp. field stage 4 larvae aftermalathion exposure.

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R2

= 0.2689

1.45

1.5

1.55

1.6

1.65

1.7

1.75

0 5 10 15 20

Concentration (ppm)

Averagelive

rsize(mm)

Figure 7. Liver size ofBufo sp. field stage 4 larvae exposed to malathion at

varying concentrations.

In addition, abnormal behaviors including circular swimming pattern

instead of a straight trajectory, decreased frequency of swimming and tail

twitching were observed with tadpoles exposed to malathion. Circular

swimming pattern were found to be associated with axis bending as observed

in the tadpoles exposed with malathion.

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DISCUSSION

Results of this experiment showed that concentrations at 20 ppm is

already lethal to Bufo sp. larvae in field stage four since 100% mortality was

attained in a short time of exposure of 48 hours. Bufo sp. larvae were found to

be sensitive with the slight difference of the concentration from 15 ppm to 20

ppm. The result showed that Bufo sp. larvae are quite sensitive to malathion.

Not all Bufo species, however, offer the same sensitivity. For instance,

embryos of arenarum were found to be quite resistant to malathion with an

LC505d of 42 ppm (Rosenbaum et al. 1988).

Mortality caused by exposure to malathion can be attributed to its

AChE inhibitory effect and other mechanisms such as increase susceptibility

to microbial infections due to decreased immunocompetency due to exposure

to malathion (Kiesecker, 2002).

The LC50 values were found to decrease with increasing length of

exposure to malathion owing to the bioaccumulation of malathion in exposed

tadpoles. This was so because exposure to high concentrations such as 20

ppm already caused death after 48 hours but in lower concentrations, deaths

were more apparent as length of exposure was increased.

At lower concentrations, anurans were found to be capable of

bioaccumulation of OP pesticides to levels considered lethal to other

organisms (Hall and Kolbe, 1980). This can be related to their reduce

dependence on pulmonary respiration, making them relatively resistant to

AChE (Acetylcholine esterase) inhibition. AChE inhibition often results to

respiratory paralysis, bronchoconstriction and increase bronchial excretions.

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Effects of toxicity were manifested through morphological abnormalities

such as curved tail, bent body axis (head) and bulging at the lower right

abdominal region. This result was consistent with the study conducted by

Chemotti et al. (2006) onXenopus laevis after a 3-day exposure to malathion.

The maximum average degree (65.6 degrees) of curvature of tail occurred in

the larvae exposed to 0.25 ppm malathion. In the experiment, however, the

maximum average degree (12.6 degrees) of tail curvature of Bufo sp. was

observed in 15 ppm. In a previous study, Pawar et al. (1983) also found body

curvature ofMicrohyla ornata tadpoles when exposed to 510 ppm malathion.

In addition, Pawar et al. (1983) observed unusual behaviors including loss of

balance, circular pattern of swimming, and decrease in activity. Similar results

were obtained in this study.

The mechanisms by which the pesticide causes axis deformation are

not well understood. Chemotti et al. (2006) argued, however, that bending of

axis may be related to the integrity of the extracellular matrix making up the

notochord. Snawder and Chambers (1993) showed that malathion reduces

the number of extracellular collagen by reducing the amount of ascorbic acid

and hydroxyproline levels necessary for the formation of collagens triple helix.

In this study, the direction of tail bending was found to be dorsal suggesting

that bending was possibly caused by deformities of the notochord and not by

contractions of the tail muscles. This was not tested, however, in this study.

Head bending was not observed in the group exposed to 20 ppm.

Unlike that of tail curvature which was observed after 48 hours, axis deformity

in the head takes time to develop. This means malathion must first be

bioaccumulated in the body of the tadpoles to induce axis deformation at the

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head region. This requires exposure at lower concentrations than that which

can cause lethality at short exposure.

Liver toxicity of malathion was not clearly defined in the experiment

since it was also found that liver size in the control group had no significant

difference to those in the experiment group. Liver toxicity of malathion has

limited literature. However, studies have shown that malathion, when

absorbed, is degraded in the liver and produces products more potent than

the original compound. It is therefore, recommended in this study to

investigate further the effects of malathion on liver size and function of Bufo

sp. larvae or other anuran species.

Organ displacement was also considered in the presence of bulging of

the lower right abdominal region since the opposite region was found to be

depressed.

Abnormal behaviors were also manifested by the exposed organisms.

These abnormalities in behavior include loss of balance, swimming in a

circular pattern and constant twitching of the tail during swimming and at the

stationary state. Loss of balance and swimming in a circular pattern were

more prominent in tadpoles which developed axis deformation. The direction

of swimming also tends to go to the direction of the bend. It was therefore

believed that these behaviors are consequences from the bending of body

axis. Muscle twitching in the tail, on the other hand, was considered to be the

caused by the anticholinesterase property of the organophosphate, malathion.

Ragnarsdottir (2000), as well as a couple of people studying

organophosphate toxicities, had confirmed that continuous muscle contraction

is a result of the inhibition of acetylcholinesterase (Webb et al., 2006).

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Abnormal behaviors were usually followed by death as observed in the

current study. In the natural environment, these effects can have

consequences on the organisms survival. Tadpoles that develop a bent body

axis reduce its ability for normal locomotion, which in turn can limit the ability

to reach food sources and increase the risks of predation and desiccation.

CONCLUSION

The calculated values of LC50 from 9th-12th day ranged from 6 to 13

ppm in a decreasing pattern, showing that levels of toxicity of Bufo sp. field

stage IV larva with malathion increases with constant exposure. Mortality in

Bufo sp. larvae exposed to malathion was dosage dependent (R2 = 0.9194).

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Exposure also produced abnormalities in morphology including: axis

deformities in the head and tail and the presence of a bulge on the lower right

abdominal region. Axis deformities include: tail curvature and head bending

with a golf-like pattern. Tail curvature and head bending was dosage

dependent (R2 = 0.9431 and 0.7876, respectfully) signifying a high influence

of malathion in larval deformities. The degrees of curvature of tail significantly

differed (P


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studies must be pursued regarding the toxicity of malathion and other

organophosphates on other anuran species.

One specific malformation observed in the experiment was liver

edema, however the mechanism on how malathion can cause such result was

not clearly defined. Therefore, further investigations on liver edema as a

response to malathion exposure should be considered.

This study was designed in the lab to solely define the acute toxicity

effects of malathion only. The experiments done definitely did not replicate the

natural environment of the Bufo sp. larvae. Therefore, the effects observed in

the lab may vary with those present in the real environment due to other

environmental factors such as presence or absence of other chemicals,

presence of predators, and etc. It is recommended, therefore, that further

studies should also consider designing set ups that could replicate the true

environment of Bufo sp. or other anuran species and the effects of other

environmental factors (if factors increase or decrease the degree of response

towards organophosphates).

ACKNOWLEDGEMENT

I would like to give my sincere gratitude to my adviser, Prof. Florence

Evacitas, for her patience and guidance in the laboratory and in making the

manuscript. I would also like to thank Miss Ruby Caminade and especially Mr.

Tristan Arvin Jain, who generously gave their assistance in the field and in the

laboratory. Lastly, I would also thank the University of the Philippines in the

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Visayas Cebu College for the equipment and facilities and as the place to

conduct my laboratory work.

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